Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.     

酵母双杂交系统筛选与RapGAP相互作用的蛋白质

目的筛选与Rap GAP相互作用的蛋白质,为进一步研究人源Rap1GAP介导的信号转导通路、揭示其与肿瘤的关系提供实验依据。方法选用与Rap1GAP同源的来自美丽线虫的Rap GAP作为饵蛋白,以来源于美丽线虫的c DNA文库作为靶蛋白,应用p PC97、p PC86组成的酵母双杂交系统筛选c DNA文库中与Rap GAP相互作用的蛋白质。结果通过营养缺陷平板(-LTH)筛选出63个拟似阳性菌落。经过Lac Z鉴定,19个菌落为阳性,其中7个为强阳性。提取来自19个酵母菌落中的重组DNA,经PCR扩增,12个菌落出现阳性结果。将该19个重组DNA分别电转化入DH5α细菌,涂板培养后,每板挑取4~10个克隆,通过Sal I和Not I双酶切鉴定进行阳性克隆筛选。将阳性克隆的重组DNA进行序列测定。测序结果与Gen Bank比较,其中4个克隆的DNA片段为Y39b6a基因片段、2个为Rap GAP、1个为苯丙氨酸-4-羟化酶、1个为细胞色素C氧化酶,还有1个DNA片段编码美丽线虫特有的小分子蛋白的基因片段,其余11个DNA片段不编码已知蛋白质。结论初步筛选出与Rap GAP相互作用的蛋白质,特别是其中有2个克隆为Rap GAP,提示Rap GAP可能以二聚体的方式存在。     

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

Properties and submitochondrial localization of pig and rat renal phosphate-activated glutaminase

Two pools ofphosphate-activated glutaminase (PAG) were separated from pig and ratrenal mitochondria. The partition of enzyme activity corresponded withthat of the immunoreactivity and also with the postembedding immunogoldlabeling of PAG, which was associated partly with the inner membraneand partly with the matrix. The outer membrane was not labeled. PAG inintact mitochondria showed enzymatic characteristics that were similarto that of the membrane fraction and also mimicked that of thepolymerized form of purified pig renal PAG. PAG in the soluble fractionshowed properties similar to that of the monomeric form of purifiedenzyme. It is indicated that the pool of PAG localized inside the innermitochondrial membrane is dormant due to the presence of highconcentrations of the inhibitor glutamate. Thus the enzymaticallyactive PAG is assumed to be localized on the outer face of the innermitochondrial membrane. The activity of this pool of PAG appears to beregulated by compounds in the cytosol, of which glutamate may be most important.

     

Bayesian analysis of wildlife age-at-harvest data

SUMMARY: State and federal natural resource management agencies often collect age-structured harvest data. These data represent finite realizations of stochastic demographic and sampling processes and have long been used by biologists to infer population trends. However, different sources of data have been combined in ad hoc ways and these methods usually failed to incorporate sampling error. In this article, we propose a "hidden process" (or state-space) model for estimating abundance, survival, recovery rate, and recruitment from age-at-harvest data that incorporate both demographic and sampling stochasticity. To this end, a likelihood for age-at-harvest data is developed by embedding a population dynamics model within a model for the sampling process. Under this framework, the identification of abundance parameters can be achieved by conducting a joint analysis with an auxiliary data set. We illustrate this approach by conducting a Bayesian analysis of age-at-harvest and mark-recovery data from black bears (Ursus americanus) in Pennsylvania. Using a set of reasonable prior distributions, we demonstrate a substantial increase in precision when posterior summaries of abundance are compared to a bias-corrected Lincoln-Petersen estimator. Because demographic processes link consecutive abundance estimates, we also obtain a more realistic biological picture of annual changes in abundance. Because age-at-harvest data are often readily obtained, we argue that this type of analysis provides a valuable strategy for wildlife population monitoring.     

On the effect of misclassification on bias of perfectly measured covariates in regression

This note clarifies under what conditions a naive analysis using a misclassified predictor will induce bias for the regression coefficients of other perfectly measured predictors in the model. An apparent discrepancy between some previous results and a result for measurement error of a continuous variable in linear regression is resolved. We show that similar to the linear setting, misclassification (even when not related to the other predictors) induces bias in the coefficients of the perfectly measured predictors, unless the misclassified variable and the perfectly measured predictors are independent. Conditional and asymptotic biases are discussed in the case of linear regression, and explored numerically for an example relating birth weight to the weight and smoking status of the mother.